Secondary‐type mutations do not impact prognosis in acute myelogenous leukemia <scp>AML</scp> with mutated <i>NPM1</i>
Authors: Martha F. Wright, Olga Pozdnyakova, Robert P. Hasserjian, Nidhi Aggarwal, Aaron C. Shaver, Olga K. Weinberg, Rebecca Irlmeier, Tatsuki Koyama, Adam C. Seegmiller, Stephen A. Strickland, Emily F. Mason
Journal: American Journal of Hematology (2022)

The 2022 update to the World Health Organization (WHO) classification1 revised the category of acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) to "AML, myelodysplasia-related," which now includes cases harboring "secondary-type" mutations (STM) in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and/or STAG2. Furthermore, the recent 2022 International Consensus Classification (ICC) of Myeloid Neoplasms and Acute Leukemia2 divided AML-MRC into two new categories, AML with myelodysplasia-related cytogenetic abnormalities and AML with myelodysplasia-related gene mutations. The latter includes AML with STM or mutations in RUNX1. These changes stem from studies examining the genetic ontogeny of AML, which identified STM as being highly associated with a history of myelodysplastic syndrome (MDS) or an MDS-like clinical presentation and with poor outcomes, even in patients with de novo disease.1-3 In contrast to myelodysplasia-related AML (MR-AML), AML with mutated NPM1 (NPM1+ AML) is characterized by de novo presentation and favorable outcomes.4, 5 However, NPM1+ AML may show features of myelodysplasia-related disease, and classification as NPM1+ AML or MR-AML has important prognostic and therapeutic implications. The presence of morphologic multilineage dysplasia in NPM1+ AML has no impact on patient outcome,6 while the presence of adverse risk cytogenetic abnormalities defining for MR-AML is associated with adverse prognosis in the context of an NPM1 mutation.7 Accordingly, the 2022 updated ELN AML guidelines now include NPM1+ AML with adverse cytogenetic abnormalities in the adverse risk group.8 However, the prognostic effect of STM in NPM1+ AML remains unclear. Here, we show that STM have no impact on patient outcomes in NPM1+ AML. A multi-institutional cohort of 257 cases of de novo NPM1+ AML with accompanying cytogenetic and next-generation sequencing data were evaluated (Table S1; Supplemental Methods). STM were present in 41 patients (16%) (Figure 1A; Table S2). The majority of STM occurred in SRSF2 (n = 27; 66% of STM); additional STMs occurred in ASXL1 (n = 7; 17% of STM), STAG2 (n = 5; 12% of STM), SF3B1 (n = 4; 10% of STM), BCOR (n = 1; 2% of STM), and EZH2 (n = 1; 2% of STM). No mutations were identified in U2AF1 or ZRSR2. Four patients had co-occurring SRSF2 and ASXL1 mutations. The STAG2 gene was evaluated in 132 patients; all other STM genes were evaluated in all patients. In 83% of patients, the STM VAF was greater than or equal to the NPM1 VAF (Figure 1B). Patients with STMs were more commonly male (68% male vs. 43% male; p = .003), were older at diagnosis (68.3 vs. 63.1 years; p = .003), and showed a lower platelet count at diagnosis (median 38.5 × 109/L vs. 63.5 × 109/L, p = .003). TET2 or IDH1/IDH2 mutations were significantly more likely to co-occur with STM (32/41 patients with STM vs. 120/216 patients without STM; p = .009) (Figure 1C), while DNMT3A mutations were significantly less likely to co-occur with STM (6/41 patients with STM vs.103/216 patients without STM; p < .001). FLT3-ITD mutations were more frequent in patients without STMs; however, this difference was not statistically significant (p = .06). Morphologic dysplasia was evaluated in 66 cases (see Supplemental Methods). The majority of cases (88%) showed some degree of morphologic dysplasia in at least one lineage, while 20% of cases showed multilineage dysplasia. In patients with (n = 13) and without (n = 53) STM, no difference was seen in the percentage of cases showing any dysplasia (77% vs. 91%; p = .19) or multilineage dysplasia (15% vs. 20%; p = 1). Clinical follow-up data were available for 248 patients. Median follow-up duration was 25.9 months. The majority of patients received standard induction therapy (n = 184; 74%). Although a lower percentage of patients with STM received induction therapy (62%) compared to patients without STM (77%), possibly related to the older median age of patients with STM, the difference was not statistically significant. In all, 90 (36%) patients underwent stem cell transplant (SCT); no significant difference was seen in the frequency of SCT in patients with (10/39; 26%) and without (80/173; 38%) STM. Among patients treated with induction therapy, the overall rates of primary induction failure (PIF) and complete remission with incomplete count recovery (CRi) were 11% and 13%, respectively. For patients with and without STM, there was no significant difference in rates of PIF (17% vs. 11%) or CRi (11% vs. 14%). For patients receiving induction chemotherapy, there was no significant difference in event free survival (EFS) between patients with STM (median 15.2 months) and patients without STM (median 14.8 months) (Figure 1D). Similarly, there was no significant difference in overall survival (OS) between patients with STM (median 22.9 months) and patients without STM (median 31.4 months) (Figure 1E). The 2022 updated ICC and ELN guidelines also include RUNX1 mutations as STM. Two patients in our NPM1+ AML cohort harbored RUNX1 co-mutations; one received induction chemotherapy. When EFS and OS analyses were repeated with the single RUNX1-mutated patient included in the STM group, no significant difference was seen in EFS or OS between the two groups (data not shown). STM include mutations in ASXL1, which are associated with adverse prognosis in AML as a whole. However, in this cohort, there was no difference in EFS or OS in patients with ASXL1 mutations (n = 5) and patients with other STM (n = 19) (Figure S1), although these groups are small. The frequency of FLT3-ITD mutations was higher in patients without STM, and FLT3-ITD mutations were associated with shorter EFS (9.9 months vs. 20.6 months for patients without FLT3-ITD; p = .008) and shorter OS (20.5 months vs. 79.4 months for patients without FLT3-ITD; p = .001) in the cohort overall (Figure S2A,B). Furthermore, patients with STM were significantly older at diagnosis than patients without STM. Therefore, multivariable Cox regression analyses were performed to evaluate whether these variables could be masking the effect of STM on outcomes (Table S3). In multivariable analysis, FLT3-ITD significantly impacted EFS and OS, while age at diagnosis significantly impacted OS. However, when accounting for the negative effects of these variables, the presence of STM continued to show no significant association with EFS or OS (Figure S2C,D). Finally, patients with NPM1+ AML with STM treated with induction therapy were compared with a cohort of 44 patients with MR-AML who received cytotoxic induction chemotherapy (see Supplemental Methods). There was no significant difference in the median age at diagnosis for these two groups (median 65.1 years for intensively treated patients with NPM1+ AML with STM vs. median 63.5 years for intensively treated patients with MR-AML; p = .98). Patients with MR-AML showed a higher PIF rate (18/44; 41%) than patients with NPM1+ AML with STM (4/24; 17%), although the difference was not statistically significant (p = .06). Notably, among MR-AML patients who achieved remission, 11/26 (42%) required 2 cycles of induction to do so, while no patients with NPM1+ AML with STM required 2 induction cycles to achieve remission. Furthermore, patients with MR-AML showed a significantly shorter median OS compared to patients with NPM1+ AML with STM (7.6 months vs. 22.9 months; p = .038) (Figure 1F). We show for the first time that STM occur in a minor but distinct subset (16%) of NPM1+ AML and are associated with older age, male sex, lower platelet count at diagnosis, and TET2 or IDH1/IDH2 co-mutations, but have no impact on morphologic dysplasia, PIF rate, CRi rate, EFS, or OS. SRSF2 mutations accounted for the majority of STM (27/41; 66%) in this study, occurring in 10.8% of our NPM1+ AML cohort overall. Non-SRSF2 STM were less common in our cohort, each occurring in <4% of tested patients. STM include mutations in four splicing factor genes, including SRSF2 as well as SF3B1, U2AF1, and ZRSR2. It is unclear why SRSF2, specifically, is mutated more commonly than other splicing factor genes in NPM1+ AML, but a similar finding has been seen in MDS, where SRSF2 mutations, specifically, are more likely to co-occur with IDH1 mutations.9 These data suggest that SRSF2-mutant disease may be biologically distinct from cases harboring mutations in other splicing factor genes. Cappelli et al.10 examined a cohort of NPM1+ AML patients who achieved complete molecular remission (CMR), with absence of NPM1-mutant transcripts, and showed that a significant subset (46%) showed persistence of clonal hematopoiesis at CMR, consistent with the idea that NPM1 mutations frequently occur in the context of pre-existing clonal hematopoiesis. Indeed, in our cohort, the NPM1 VAF was less than that of the STM in 83% of patients. Cappelli et al., found that, in NPM1+ AML patients achieving CMR, SRSF2 mutations behaved similarly to DNMT3A, TET2, and ASXL1 mutations in that the persistence of these mutations at CMR had no adverse effect on outcome. Taken together, our data suggest that the presence of SRSF2 or ASXL1 mutations, whether at diagnosis or at the time of CMR, should not be considered an adverse prognostic factor in patients with NPM1+ AML. This is a retrospective study analyzing NPM1+ AML cohorts from four institutions, which allowed for analysis of a relatively rare subset of patients. We performed outcome measurements only on patients treated with intensive induction chemotherapy, to further standardize the cohort. Although recent studies have shown that STM hold prognostic significance for some patients with de novo AML, our results suggest that in NPM1+ AML, the NPM1 mutation drives disease biology and patient outcome. These results are particularly relevant given the recently updated WHO1 and International Consensus2 Classifications, which include STM as defining for MR-AML. Based on our results, we suggest that STM must be considered in the context of other clinicopathologic, cytogenetic, and molecular features, and that patients with NPM1+ AML with STM should be considered within the NPM1+ AML category for the purposes of prognostication and treatment. Contribution: Martha F. Wright and Emily F. Mason designed the study, collected data, performed data analysis, and prepared the manuscript. Olga Pozdnyakova, Robert P. Hasserjian, Nidhi Aggarwal, Aaron C. Shaver, Olga K. Weinberg, Tatsuki Koyama, Rebecca Irlmeier, Adam C. Seegmiller, Stephen A. Strickland contributed to data collection, data analysis, and manuscript review. The authors have no conflicts of interest to declare. The data that support the findings of this study are available from the corresponding author upon reasonable request. TABLE S1. Clinicopathologic characteristics of patients with NPM1+ AML, with and without secondary-type mutations TABLE S2. Secondary-type mutations and co-mutations in patients with NPM1+ AML TABLE S3. Multivariable analyses for factors associated with event-free and overall survival FIGURE S1. Kaplan–Meier analysis in patients with NPM1+ AML with ASXL1 mutations and patients with other secondary-type mutations. (A) There was no significant difference in EFS between patients with ASXL1 mutations (median 19.4 months) and patients with other STM (median 9.9 months). (B) There was no significant difference in OS between patients with ASXL1 mutations (median undefined) and other STM (median 22.9 months). FIGURE S2. Kaplan–Meier analysis of patients with NPM1+ AML with and without secondary mutations and FLT3-ITD. (A-B) The presence of a FLT3-ITD was significantly associated with shorter EFS (A) and OS (B) in the cohort of NPM1+ AML as a whole (EFS 9.9 months and OS 20.5 months in patients with FLT3-ITD versus EFS 20.6 months and OS 79.4 months for patients without FLT3-ITD). (C-D) Similar results were seen when examining patients with and without STM. Median EFS (C) for each group: STM-/FLT3-ITD-: 22.4 months; STM-/FLT-ITD+: 10.0 months; STM+/FLT3-ITD-: 19.4 months; STM+/FLT3-ITD+: 9.9 months. Median OS (D) for each group: STM-/FLT3-ITD-: 79.4 months; STM-/FLT-ITD+: 20.5 months; STM+/FLT3-ITD-: 37.8 months; STM+/FLT3-ITD+: 16.9 months Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.